This application claims the benefit of U.S. Provisional Application 61/139,852, filed Dec. 22, 2008 and is incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with United States Government support under Contract No. 04-03-CA-70224 awarded by the Department of Energy and Contract No. DE-AC36-08G028308 between the United States Department of Energy and the Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory. The U.S. Government has certain rights in this invention.

FIELD OF INVENTION

The invention relates to the fields of microbiology and fermentation. More specifically, development of Zymomonas strains with improved xylose utilization under stress fermentation conditions is described.

BACKGROUND OF INVENTION

Production of ethanol by microorganisms provides an alternative energy source to fossil fuels and is therefore an important area of current research. It is desirable that microorganisms producing ethanol, as well as other useful products, be capable of using xylose as a carbon source since xylose is the major pentose in hydrolyzed lignocellulosic materials, and therefore can provide an abundantly available, low cost carbon substrate. Zymomonas mobilis and other bacterial ethanologens which do not naturally utilize xylose may be genetically engineered for xylose utilization by introduction of genes encoding 1) xylose isomerase, which catalyses the conversion of xylose to xylulose; 2) xylulokinase, which phosphorylates xylulose to form xylulose 5-phosphate; 3) transketolase; and 4) transaldolase.

There has been success in engineering Z. mobilis cells for xylose metabolism (U.S. Pat. No. 5,514,583, U.S. Pat. No. 5,712,133, U.S. Pat. No. 6,566,107, WO 95/28476, Feldmann et al. (1992) Appl Microbiol Biotechnol 38: 354-361, Zhang et al. (1995) Science 267:240-243), as well as a Zymobacter palmae strain (Yanase et al. (2007) Appl. Environ. Mirobiol. 73:2592-2599). However, typically the engineered strains do not grow and produce ethanol as well on xylose as on glucose. Strains engineered for xylose utilization have been adapted by serial passage on xylose medium, resulting in strains with improved xylose utilization as described in United States Patent Application 20030162271 and commonly owned and co-pending US Patent Application Publication No. US 2008-0286870 A1. It has been shown that these improvements were the result of selection for altered sequences for improved expression of the pGAP promoter regulating expression of the xylose isomerase gene. Those sequences and methods for their use in improved expression of transgenes in Z. mobilis are disclosed in commonly owned and co-pending United States Patent Application Publication Nos. US2009-0246876 A1 and US2009-0246846 A1.

It is desired to use cellulosic hydrolysates as a renewable source of sugars for fermentation media for production of ethanol by biocatalysts. Cellulosic hydrolysates, which are generally produced from biomass by pretreatment and saccharification, typically contain substances that are detrimental to biocatalyst growth and production. For example, acetate is a common product present in cellulosic hydrolysates which has been shown to be inhibitory to Z. mobilis at concentrations routinely found in hydrolysate (Ranatunga et al. (1997) Applied Biochemistry and Biotechnology 67:185-198).

There remains a need for strains of Zymomonas, and other bacterial ethanologens, which have maximized xylose utilization in the presence of stresses imposed by impure sugar sources produced through saccharification of biomass.

SUMMARY

The invention provides a method for obtaining xylose-utilizing Zymomonas strains that have improved xylose utilization under stress fermentation conditions as well as strains of Zymomonas produced using this method.

Accordingly, the invention provides an isolated improved xylose-utilizing Zymomonas strain obtained by a method comprising:
a) providing xylose-utilizing Zymomonas cells;
b) continuously growing the xylose-utilizing Zymomonas cells of (a) in a feeding growth medium comprising xylose, whereby a culture comprising ethanol is produced;
c) adding to the culture made in (b) an amount of ammonia and acetic acid or ammonium acetate, whereby a stress culture comprising ethanol and ammonium acetate is produced;
d) continuously growing the stress culture made in (c), whereby improved xylose-utilizing Zymomonas cells are produced, wherein the improvement in the cells is increased conversion of xylose to ethanol in the presence of ammonium acetate compared to the Zymomonas cells of (a);
e) isolating one or more cells from the improved culture made in (d); and
f) growing the one or more improved cells to make the strain.

In a further embodiment the invention is a stress fermentation adapted xylose-utilizing Zymomonas strain that uses at least about 70% of available xylose in a mixed sugars medium, e.g. a medium containing 60 g/L xylose, 80 g/L glucose, 9.54 g/L acetate, with pH adjusted to 5.8, when starting at a cell density of 0.1 OD600 nm, as compared to the corresponding non-adapted strain utilization of about 18% of available xylose.

In a further embodiment, the invention provides a method of producing ethanol comprising:
a) providing an improved stress fermentation adapted xylose-utilizing Zymomonas strain as described above;
b) contacting the strain of a) with a fermentation medium under suitable fermentation conditions wherein ethanol is produced; and
c) optionally isolating the ethanol.

The stress fermentation adapted cells or strains are provided by the adaptation procedures herein, specifically with reference to adaptation to medium comprising ethanol and ammonium acetate.

DETAILED DESCRIPTION

The present invention describes a method for producing and isolating Zymomonas cells (grown to be strains) that have improved utilization of xylose under stress fermentation conditions. The Zymomonas cells to which the method is applied are xylose-utilizing cells, which, according to the invention, are continuously grown under conditions of ammonium acetate and ethanol stress to produce stress fermentation adapted xylose-utilizing Zymomonas strains. The present invention is also directed to stress fermentation adapted xylose-utilizing Zymomonas strains that are isolated using the present method, and which utilize a higher percent of input xylose under stress fermentation conditions as compared to the cells of the same strain prior to the continuous growth process. The stress fermentation adapted strains may be used in a process for producing ethanol by fermenting sugars. Ethanol produced by the present stress fermentation adapted Zymomonas strains may be used as an alternative energy source to fossil fuels.

The following abbreviations and definitions will be used for the interpretation of the specification and the claims.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

As used herein “continuously growing” refers to growing with input of new medium and exit flow of effluent such that cells may continue to grow and produce product.

As used herein “stress culture” refers to a culture that includes substances in the medium that cause stress to a biocatalyst used in the culture. Stress of a biocatalyst may be recognized as reduced growth rate, reduced product production, reduced carbohydrate utilization, or other difficulty as compared to function of a biocatalyst used in fermentation without the substances causing stress. Of particular interest are stressers to Zymomonas strains that affect sugar utilization for ethanol production. Such stressers include the presence of acetate, ammonia, and ethanol. Of further interest is the effect such stressers have on xylose utilization for the production of ethanol.

As used herein “xylose-utilizing Zymomona cell(s)” refers to a cell or cells of a strain that are genetically engineered to express enzymes conferring the ability to use xylose as a carbohydrate source for fermentation.

As used herein “corresponding non-adapted strain” refers to the original xylose-utilizing Zymomonas strain that is a strain from which improved strains are produced using the stress adaptation process disclosed herein.

As used herein “feeding growth medium” refers to the medium that is added into the continuous culture vessel.

As used herein “hydrolysate of biomass” and “cellulosic hydrolysate” refer to a product produced from biomass, which is cellulosic material, typically through pretreatment and saccharification processes. Fermentable sugars are present in the hydrolysate, as well as other products.

Increased Xylose Utilization

Applicants have found that xylose-utilizing Zymomonas strains may be made to utilize increased amounts of xylose under stress fermentation conditions by adapting the strains through a process involving continuous growth under stress conditions. Increase in xylose utilization is compared to xylose utilization by a xylose-utilizing Zymomonas strain that has not undergone adaptation processes as described herein. Stress conditions used herein during adaptation of Zymomonas strains provide similar stress conditions to those present when growing Zymomonas strains in medium comprising hydrolysate of biomass. Thus the present adapted strains may have increased xylose utilization when grown in medium comprising hydrolysate of biomass, thereby providing more efficient growth and product formation.

Any strain of Zymomonas that is able to utilize xylose as a carbon source may be an original or starting strain provided for adaption and used in the present method for preparing the stress fermentation adapted xylose-utilizing Zymomonas strains that exhibit the improved xylose utilization in accordance with the present invention. Strains of Zymomonas, such as Z. mobilis, that have been engineered for xylose fermentation to ethanol are particularly useful. Endogenous genes may provide part of the metabolic pathway, or may be altered by any known genetic manipulation technique to provide a protein with enzyme activity useful for xylose metabolism. For example, the endogenous transketolase may complement other introduced enzyme activities in creating a xylose utilization pathway. Typically four genes may be introduced into a Zymomonas strain, such as Z mobilis, for expression of four enzymes involved in xylose metabolism as described in U.S. Pat. No. 5,514,583, which is herein incorporated by reference. These include genes encoding xylose isomerase, which catalyzes the conversion of xylose to xylulose and xylulokinase, which phosphorylates xylulose to form xylulose 5-phosphate. In addition, transketolase and transaldolase, two enzymes of the pentose phosphate pathway, convert xylulose 5-phosphate to intermediates that couple pentose metabolism to the glycolytic Entner-Douderoff pathway permitting the metabolism of xylose to ethanol. DNA sequences encoding these enzymes may be obtained from any of numerous microorganisms that are able to metabolize xylose, such as enteric bacteria, and some yeasts and fungi. Sources for the coding regions include Xanthomonas, Klebsiella, Escherichia, Rhodobacter, Flavobacterium, Acetobacter, Gluconobacter, Rhizobium, Agrobacterium, Salmonella, Pseudomonads, and Zymomonas. Particularly useful are the coding regions of E. coli.

The encoding DNA sequences are operably linked to promoters that are expressed in Z. mobilis cells such as the promoters of Z. mobilis glyceraldehyde-3-phosphate dehydrogenase (GAP promoter), and Z. mobilis enolase (ENO promoter). The coding regions may individually be expressed from promoters, or two or more coding regions may be joined in an operon with expression from the same promoter. The resulting chimeric genes may be introduced into Zymomonas and maintained on a plasmid, or integrated into the genome using, for example, homologous recombination, site-directed integration, or random integration. Xylose-utilizing strains that are of particular use include ZM4(pZB5) (described in U.S. Pat. No. 5,514,583, U.S. Pat. No. 6,566,107, and US55712133, and incorporated by reference herein), 8b (US 20030162271; Mohagheghi et al., (2004) Biotechnol. Lett. 25; 321-325), as well as ZW658 (ATTCC # PTA-7858), ZW800, ZW801-4, ZW801-5, and ZW801-6 (described in commonly owned and co-pending US Patent Application Publication #US 2008-0286870 A1, which is herein incorporated by reference).

Zymomonas strains that are additionally engineered to utilize other sugars that are not natural substrates, may also be used in the present process. An example is a strain of Z. mobilis engineered for arabinose utilization as described in U.S. Pat. No. 5,843,760, which is herein incorporated by reference.

Adaptation Method

In the present method, a xylose-utilizing strain of Zymomonas (a starting or original strain as described above) is continuously grown in medium comprising xylose under stress fermentation conditions. The medium may contain xylose as the only sugar, or it may contain a mixture of xylose and other sugars such as glucose. Preferred is a high sugars concentration in the medium, for example at least about 50 g/L each of xylose and glucose. There may be more of either or both sugars.

The original xylose-utilizing strain of Zymomonas is first grown continuously without stress conditions and ethanol is produced. Feeding growth medium is added at a dilution rate to maintain the continuous culture. Typically the culture is allowed to stabilize, which may take about 9-12 days, although some cultures may take longer to stabilize. Stabilization is with respect to OD600, sugar utilization, and ethanol production as measured in the effluent from the fermentor. Ethanol in the stabilized culture may be produced to a level of about 18 g/L, 22 g/L, 40 g/L or higher. Additional components are then added to the fermentation medium that cause stress conditions for metabolism of the Zymomonas cells. These components may be added just after stabilization of the culture, or after the culture is fermented under stable conditions for a further period of time. The components that may be added as stressers include ammonia and acetic acid, which results in medium containing ammonium acetate, or ammonium acetate. The presence of ammonium acetate and ethanol in the fermentation medium applies stress to the Zymomonas cells, which typically causes a decrease in OD600, decrease in xylose utilization, and decrease in ethanol production. The dilution rate may be increased or decreased to manage the OD600, xylose utilization, and ethanol concentration. The culture under stress conditions is continuously grown. More ammonia and acetic acid or ammonium acetate may be added at least one or more times during the continuous fermentation of the stress culture. Addition may be more than one time; such as in stages to gradually increase the ammonium acetate concentration in the fermentation medium.

The ammonia and acetic acid may be added as ammonium acetate initially to achieve concentrations of about 24 mM ammonium acetate or about 48 mM ammonium acetate. Ammonia and acetic acid or ammonium acetate may be added in one or more increments to reach concentrations that are between about 64 mM to 210 mM of ammonium acetate. Typically, ammonia and acetic acid or ammonium acetate are added in four or more steps over time to gradually increase the concentration in the fermentation medium of the stress culture. Ethanol in the stress culture is produced by the Zymomonas cells and may vary depending on the production rate, typically ethanol is between about 13 g/L and 54 g/L. The OD600 may vary, and is typically between about 1.5 and about 4.8.

Following a total continuous fermentation period of about two months or more, strains of adapted Zymomonas may be isolated from the stress culture. Samples may be taken from the culture and streaked onto plates to isolate colonies, or grown in culture and then streaked onto plates to isolate colonies. Cells from individual colonies are tested for the capability of isolated cells or strains grown from the cells to utilize xylose and produce ethanol in medium containing high sugar concentration, ammonia and acetate. Ethanol is produced in the cultures by the adapted Zymomonas cells. Among the isolated strains tested, strains are identified that show increased xylose utilization and increased ethanol production as compared to the original or starting xylose-utilizing strain prior to adaptation. One skilled in the art is well aware that there will be variation in the amount of xylose utilized and ethanol produced in different isolated strains. However, strains that have increased xylose utilization and ethanol production will be readily identifiable and recognized among isolates from the stress fermentation cultures.

Adapted Strains

Disclosed herein are stress fermentation adapted xylose-utilizing Zymomonas strains with improved xylose utilization. These strains may be characterized by an increase in utilization of xylose of at least about 12% as compared to the corresponding but non-adapted strain when grown under stress conditions wherein fermentation medium contains ethanol, ammonium acetate and high sugar concentration. Adapted strains may use at least about 12%, 17%, 20%, 25%, or 30% more xylose. Strains of xylose-utilizing Zymomonas having these characteristics may be routinely isolated using the disclosed method. The amount of xylose utilization will depend upon factors including the original strain, the conditions of fermentation, and the particular adapted strain itself.

Under one set of conditions described in Example 3 herein with media containing 60 g/L xylose, 80 g/L glucose, 9.54 g/L acetate, and 160 mM NH4OH when starting at a cell density of 0.1 OD at 600 nm, improved adapted strains may use at least about 70%, 80%, 85%, or 89% of xylose while the original strain uses about 17% of xylose. Under varied conditions, adapted strains may use at least about 40%, 45%, 50%, 55%, 60%, 65% or 70% of xylose as compared to 30% or less by strains not adapted by stress fermentation as described herein.

Adapted strains of the invention may be used for fermenting sugars to produce fermentation products such as ethanol. The strains are particularly useful for fermentation in medium containing hydrolysate of biomass which contains components providing stress fermentation conditions. Lignocellulosic biomass is typically subjected to certain processes to produce fermentable sugars. These processes may include pre-processing, pretreatment, and saccharification. Pre-processing is any action that renders the biomass more susceptible to pretreatment. Pretreatment is any processing that renders the biomass more susceptible to saccharification. Saccharification includes any processing that hydrolyzes biomass carbohydrates to fermentable sugars. Fermentable sugars include mono and oligo saccharides that can be utilized by a biocatalyst for fermentation. Any commonly known methodologies may be used for pre-processing and pretreating biomass including no pre-processing and/or no pretreatment. Commonly known saccharification techniques may be employed to produce a hydrolysate for fermentation such that sugars are available for utilization, including enzymatic hydrolysis and/or auto or chemical hydrolysis. For example, biomass may be pretreated and saccharified as described in commonly owned and co-pending US Patent Publication US20070031918A1.

Fermentation for Ethanol Production

For production of ethanol, a Zymomonas strain of the invention is contacted with medium that contains mixed sugars including xylose. When the mixed sugars concentration is high such that growth is inhibited, the medium may include sorbitol, mannitol, or mixtures thereof. Galactitol or ribitol may replace or be combined with sorbitol or mannitol. The Zymomonas grows in the medium where fermentation occurs and ethanol is produced. The fermentation is run without supplemented air, oxygen, or other gases (which may include conditions such as anaerobic, microaerobic, or microaerophilic fermentation), for at least about 24 hours, and may be run for 30 or more hours. The timing to reach maximal ethanol production is variable, depending on the fermentation conditions. Typically, if inhibitors are present in the medium, a longer fermentation period is required. The fermentations may be run at temperatures that are between about 30° C. and about 37° C., at a pH of about 4.5 to about 7.5.

The xylose-utilizing Zymomonas (such as Z. mobilis) may be grown in medium containing mixed sugars including xylose in laboratory scale fermenters, and in scaled-up fermentations where commercial quantities of ethanol are produced. Where commercial production of ethanol is desired, a variety of culture methodologies may be applied for producing ethanol utilizing the adapted strains described herein. For example, large-scale production may be run by both batch and continuous culture methodologies. A classical batch culturing method is a closed system where the composition of the medium is set at the beginning of the culture and not subjected to artificial alterations during the culturing process. Thus, at the beginning of the culturing process the medium is inoculated with the desired organism and growth or metabolic activity is permitted to occur adding nothing to the system. Typically, however, a “batch” culture is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase are often responsible for the bulk of production of end product or intermediate in some systems. Stationary or post-exponential phase production can be obtained in other systems.

A variation on the standard batch system is the Fed-Batch system. Fed-Batch culture processes are also suitable for growth of the present strains and comprise a typical batch system with the exception that the substrate is added in increments as the culture progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH and the partial pressure of waste gases such as CO2. Batch and Fed-Batch culturing methods are common and well known in the art and examples may be found in Biotechnology: A Textbook of Industrial Microbiology, Crueger, Crueger, and Brock, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36, 227, (1992), herein incorporated by reference.

Commercial production of ethanol may also be accomplished with a continuous culture. Continuous cultures are open systems where a defined culture medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous cultures generally maintain the cells at a constant high liquid phase density where cells are primarily in log phase growth. Alternatively, continuous culture may be practiced with immobilized cells where carbon and nutrients are continuously added, and valuable products, by-products or waste products are continuously removed from the cell mass. Cell immobilization may be performed using a wide range of solid supports composed of natural and/or synthetic materials as is known to one skilled in the art.

Continuous or semi-continuous culture allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by medium turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to medium being drawn off must be balanced against the cell growth rate in the culture. Methods of modulating nutrients and growth factors for continuous culture processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

Particularly suitable for ethanol production is a fermentation regime as follows. The desired adapted strain is grown in shake flasks in semi-complex medium at about 30° C. to about 37° C. with shaking at about 150 rpm in orbital shakers and then transferred to a 10 L seed fermentor containing similar medium. The seed culture is grown in the seed fermentor anaerobically until OD600 is between 3 and 6, when it is transferred to the production fermentor where the fermentation parameters are optimized for ethanol production. Typical inoculum volumes transferred from the seed tank to the production tank range from about 2% to about 20% v/v. Typical fermentation medium contains minimal medium components such as potassium phosphate (1.0-10.0 g/l), ammonium sulfate (0-2.0 g/l), magnesium sulfate (0-5.0 g/l), a complex nitrogen source such as yeast extract or soy based products (0-10 g/l). A final concentration of about 5 mM sorbitol or mannitol is present in the medium. Mixed sugars including xylose and at least one additional sugar such as glucose (or sucrose), providing a carbon source, are continually added to the fermentation vessel on depletion of the initial batched carbon source (50-200 g/l) to maximize ethanol rate and titer. Carbon source feed rates are adjusted dynamically to ensure that the culture is not accumulating glucose in excess, which could lead to build up of toxic byproducts such as acetic acid. In order to maximize yield of ethanol produced from substrate utilized, biomass growth is restricted by the amount of phosphate that is either batched initially or that is fed during the course of the fermentation. The fermentation is controlled at pH 5.0-6.0 using caustic solution (such as ammonium hydroxide, potassium hydroxide, or sodium hydroxide) and either sulfuric or phosphoric acid. The temperature of the fermentor is controlled at 30° C.-35° C. In order to minimize foaming, antifoam agents (any class-silicone based, organic based etc) are added to the vessel as needed. An antibiotic, for which there is an antibiotic resistant marker in the strain, such as kanamycin, may be used optionally to minimize contamination.

Any set of conditions described above, and additionally variations in these conditions that are well known to one skilled in the art, are suitable conditions for production of ethanol by an adapted xylose-utilizing Zymomonas strain of the invention.

Ethanol produced in the fermentation may be recovered using various methods known in the art. As a specific example, bioproduced ethanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see for example, Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process. Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the ethanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.

EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

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